Methods of screening agents, conjugates or conjugate moieties for transport by a pept2 transporter

ABSTRACT

The invention provides methods of screening agents, conjugates or conjugate moieties, linked or linkable to agents, for capacity to be transported as substrates through the PEPT2 transporter. The invention also provides methods of treatment involving delivery of agents that either alone, or as a result of linkage to a conjugate moiety, are substrates of the PEPT2 transporter. The invention also provides conjugates comprising a pharmaceutical agent which is linked to a conjugate moiety that is a substrate for a PEPT2 transporter.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 11/134,723 filed on May 20, 2005, which is a continuation of U.S.patent application Ser. No. 10/170,217 filed on Jun. 11, 2002, issued asU.S. Pat. No. 6,955,888 on Oct. 18, 2005, which claims benefit of U.S.Provisional Application No. 60/361,002 filed Mar. 1, 2002, and U.S.Provisional Application No. 60/297,732 filed on Jun. 11, 2001, each ofwhich is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Recent advances in the pharmaceutical industry have resulted in theformation of an increasing number of potential therapeutic agents.However, formulating the compounds for effective oral bioavailabilityhas proven difficult because of problems associated with uptake and highsusceptibility to metabolic enzymes.

Natural transporter proteins are involved in the uptake of variousmolecules into and/or through cells. In general, two major transportsystems exist: solute carrier-mediated systems and receptor mediatedsystems. Carrier-mediated systems use transport proteins that areanchored to the cell membrane, typically by a plurality ofmembrane-spanning loops and function by transporting their substratesvia an energy-dependent flip-flop or other mechanism, exchange and otherfacilitative or equilibrative mechanisms. Carrier-mediated transportsystems are involved in the active or non-active, facilitated transportof many important nutrients such as vitamins, sugars, and amino acids.The carrier systems result in transport into the enterocytes from bloodor lumen, and across the epithelial cell layer from lumen into blood(absorption) or blood to lumen (secretion). Carrier-mediatedtransporters are also present in organs such as the liver and kidney, inwhich the proteins are involved in the excretion or re-absorption ofcirculating compounds.

Receptor-mediated transport systems differ from the carrier-mediatedsystems in that these systems usually utilize proteins that span thecell membrane only a single time. Furthermore, substrate bindingtriggers an invagination and encapsulation process that results in theformation of various transport vesicles to carry the substrate (andsometimes other molecules) into and through the cell. This process ofmembrane deformations that result in the internalization of certainsubstrates and their subsequent targeting to certain locations in thecytoplasm is generally referred to as endocytosis.

Polar or hydrophilic compounds are typically poorly absorbed through ananimal's intestine as there is a substantial energetic penalty forpassage of such compounds across the lipid bilayers that constitutecellular membranes. Many nutrients that result from the digestion ofingested foodstuffs in animals, such as amino acids, di- andtripeptides, monosaccharides, nucleosides and water-soluble vitamins,are polar compounds whose uptake is essential to the viability of theanimal. For these substances there exist specific mechanisms for activetransport of the solute molecules across the intestinal epithelia. Thistransport is frequently energized by co-transport of ions down aconcentration gradient.

Known examples of solute carrier systems include two peptidetransporters, PEPT1 and PEPT2. The endogenous substrates for thesetransporters are small peptides consisting of two or three amino acids.These transporters function in the absorption of peptides arising fromthe digestion of dietary proteins (small intestine) and in thereabsorption of peptides present in the glomerular filtrate.

The human intestinal peptide transporter (PEPT1) and the human kidneypeptide transporter (PEPT2) exhibit about 48% identity at the amino acidlevel. Neither peptide transporter shows significant sequence identityto other known mammalian sequences—they are both about 20% identical toPHT1 and PHT2. The two transporters show some differences in therecognition of β-lactam antibiotics as substrates as well as theirmarked differences in affinity for many substrates. As such, PEPT1 is ahigh capacity, low-affinity transporter and PEPT2 is a high affinitytransporter. Both transporters accept small peptides as substrates andare driven by a transmembrane electrochemical H+ gradient.

PEPT1 and PEPT2 have been reported to show different patterns ofexpression in different human tissues. PEPT1 has been reported to beexpressed predominantly in the intestine, and also in the kidney (parsconvoluta), and liver, with small amounts of expression in the brain andpancreas. Fei et al., Nature 386:563-566 (1994) and Miyamoto et al.,Biochimica et Biophysica Acta 1305:34-38 (1996). By contrast, PEPT2 hasbeen reported to be expressed in the kidney and brain, with lowerexpression reported in the lung, liver and heart and no expressionreported in the small intestine. Liu et al., Biochimica et BiophysicaActa 1235:461-466 (1995) and Boll et al., Proc. Natl. Acad. Sci. USA93:284-289 (1996) and Saito et al., Biochimica et Biophysica Acta1280:173-177 (1996). Because of the view that PEPT1 and not PEPT2 isexpressed in the intestine, existing efforts to improve oral delivery ofdrugs via peptide transporters have focused on identifyingpharmacological agents that are, or can be modified to be, substratesfor PEPT1.

SUMMARY OF THE CLAIMED INVENTION

The present invention provides a conjugate comprising a pharmaceuticalagent which is linked to a conjugate moiety that is a substrate for aPEPT2 transporter. The conjugate has a Vmax of at least 1% of Gly-Sarfor the PEPT2 transporter. The conjugate has a greater Vmax for PEPT2than the pharmaceutical agent alone, i.e., without the conjugate moiety.

Preferably, the conjugate has a Vmax for the PEPT2 transporter of atleast 5%, more preferably at least 10%, even more preferably at least20%, still more preferably at least about 50%, and most preferably atleast 100%, respectively, of the Vmax of substrate Gly-Sar for PEPT2.

Preferably, the pharmaceutical agent without the conjugate moiety has aVmax for the PEPT2 transporter of less than 1% of the Vmax of substrateGly-Sar for PEPT2.

Preferably, the ratio of Vmax between the conjugate and Gly-Sar isgreater for the PEPT2 transporter than for the PEPT1 transporter, morepreferably the ratio for the PEPT2 transporter is at least twice, evenmore preferably at least 10 times, and most preferably at least 100times, respectively, of the ratio for the PEPT1 transporter.

The present invention also provides a method of treatment comprisingadministering a pharmacologically effective amount of the conjugate to apatient as well as a method of making a pharmaceutical compositioncomprising formulating the conjugate with a pharmaceutically acceptablecarrier.

The present invention further provides a method of screeningpharmaceutical agents, conjugates and/or conjugate moieties forpharmacological administration. The method includes providing cell(s)expressing PEPT2 transporter, contacting the cell(s) with the agent,conjugate or moiety and determining whether the agent, conjugate, ormoiety passes into and/or through the cell by way of the transporter.Preferably, the cell(s) is transfected with DNA encoding the PEPT2transporter. More preferably, the cell(s) is an oocyte injected withnucleic acid encoding the PEPT2 transporter. Even more preferably, thecell(s) exhibits no detectable PEPT1 receptor.

The invention also provides methods of manufacturing a pharmaceuticalcomposition. Such method include linking an agent to a conjugate moietyto form a conjugate wherein the conjugate is transported by the PEPT2transporter with a Vmax of at least 1% of the Vmax of the substrateGly-Sar. The conjugate is then formulated with a carrier as apharmaceutical composition.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1 shows uptake of cephradine by Caco-2 cells.

FIG. 2 shows uptake of cefadroxil by Caco-2 cells.

FIG. 3 shows F moc-amino acids.

FIG. 4 shows Boc-Alloc amino acids.

FIG. 5 shows carboxylic acid.

FIG. 6 shows Fmoc-alloc-amino acids.

FIG. 7 shows Boc-amino acids.

DEFINITIONS

The phrases “specifically binds” when referring to a protein or“specifically immunoreactive with” when referring to an antibody, refersto a binding reaction which is determinative of the presence of theprotein in the presence of a heterogeneous population of proteins andother biologics. Thus, under designated conditions, a specified ligandbinds preferentially to a particular protein and does not bind in asignificant amount to other proteins present in the sample. A moleculesuch as an antibody that specifically binds to a protein often has anassociation constant of at least 10⁶ M⁻¹ or 10⁷ M⁻¹, preferably 10⁸ M⁻¹to 10⁹ M⁻¹, and more preferably, about 10¹⁰ M⁻¹ to 10¹¹ M⁻¹ or higher.However, some substrates of transporter, PEPT1 in particular, have muchlower affinities of the order of 10-10³ M⁻¹ and yet the binding canstill be shown to be specific. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select monoclonal antibodies specifically immunoreactive with aprotein. See, e.g., Harlow and Lane (1988) “Antibodies, A LaboratoryManual”, Cold Spring Harbor Publications, New York, for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity.

A “transport protein” is a protein that has a direct or indirect role intransporting a molecule into and/or through a cell. The term includes,for example, membrane-bound proteins that recognize a substrate andeffects its entry into, or exit from a cell by a carrier-mediatedtransporter or by receptor-mediated transport. These proteins aresometimes referred to as transporter proteins. The term also includesintracellularly expressed proteins that participate in trafficking ofsubstrates through or out of a cell. The term also includes proteins orglycoproteins exposed on the surface of a cell that do not directlytransport a substrate but bind to the substrate holding it in proximityto a receptor or transporter protein that effects entry of the substrateinto or through the cell. Examples of carrier proteins include: theintestinal and liver bile acid transporters, dipeptide transporters,oligopeptide transporters, simple sugar transporters (e.g., SGLT1),phosphate transporters, monocarboxcylic acid transporters, transporterscomprising P-glycoproteins, organic anion transporters (OATP), andorganic cation transporters. Examples of receptor-mediated transportproteins include: viral receptors, immunoglobulin receptors, bacterialtoxin receptors, plant lectin receptors, bacterial adhesion receptors,vitamin transporters and cytokine growth factor receptors.

A “substrate” of a transport protein is a compound whose uptake into orpassage through a cell is facilitated by the transport protein.

The term “ligand” of a transport protein includes substrates and othercompounds that bind to the transport protein without being taken up ortransported through a cell. Some ligands by binding to the transportprotein inhibit or antagonize uptake of the substrate or passage ofsubstrates through a cell by the transport protein. Some ligands bybinding to the transport protein promote or agonize uptake or passage ofthe compound by the transport protein or another transport protein. Forexample, binding of a ligand to one transport protein can promote uptakeof a substrate by a second transport protein in proximity with the firsttransport protein.

The term “agent” is used to describe a compound that has or may have apharmacological activity. Agents include compounds that are known drugs,compounds for which pharmacological activity has been identified butwhich are undergoing further therapeutic evaluation, and compounds thatare members of collections and libraries that are to be screened for apharmacological activity.

An agent is “orally active” if it can exert a pharmalogical activitywhen administered via an oral route.

A “conjugate moiety” refers to a compound or part of a compound thatdoes not itself have pharmacological activity but which can be linked toan agent to form a conjugate that does have pharmacological activity.Typically, the agent has pharmacologic activity without the conjugatemoiety. The conjugate moiety facilitates therapeutic use of the agent bypromoting uptake of the agent via a transporter. A conjugate moiety canitself be a substrate for a transporter or can become a substrate whenlinked to a compound (e.g., valacyclovir). Thus, a conjugate moietyformed from a compound and a conjugate moiety can have higher uptakeactivity than either the compound or moiety alone.

A “pharmacological” activity means that an agent at least exhibits anactivity in a screening system that indicates that the agent is or maybe useful in the prophylaxis or treatment of a disease. The screeningsystem can be in vitro, cellular, animal or human. Agents can bedescribed as having pharmacological activity notwithstanding thatfurther testing may be required to establish actual prophylactic ortherapeutic utility in treatment of a disease.

Vmax and Km of a compound for a transporter are defined in accordancewith convention. Vmax is the number of molecules of compound transportedper second at saturating concentration of the compound. Km is theconcentration of the compound at which the compound is transported athalf of Vmax. In general, a high value of Vmax is desirable for asubstrate of a transporter. A low value of Km is desirable for transportof low concentrations of a compound, and a high value of Km is desirablefor transport of high concentrations of a compound. Vmax is affectedboth by the intrinsic turnover rate of a transporter(molecules/transporter protein) and transporter density in plasmamembrane that depends on expression level. For these reasons, theintrinsic capacity of a compound to be transported by a particulartransporter is usually expressed as the ratio Vmax of the compound/Vmaxof a control compound known to be a substrate for the transporter.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra.). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. For identifying whether a nucleic acid or polypeptide is withinthe scope of the invention, the default parameters of the BLAST programsare suitable. The BLASTN program (for nucleotide sequences) uses asdefaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4,and a comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a word length (W) of 3, an expectation (E) of10, and the BLOSUM62 scoring matrix. The TBLATN program (using proteinsequence for nucleotide sequence) uses as defaults a word length (W) of3, an expectation (E) of 10, and a BLOSUM 62 scoring matrix. (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A transporter is expressed in a particular tissue, e.g., the jejunum,when expression can be detected by mRNA analysis, protein analysis,antibody histochemistry, or functional transport assays. Typically,detectable mRNA expression is at a level of at least 0.01% of that ofbeta actin in the same tissue. Preferred transporters exhibit levels ofexpression in the desired tissue of at least 0.1, or 1 or 10% of that ofbeta actin. Conversely a transporter is not expressed in a particulartissue (e.g., the descending colon) if expression is not detectableabove experimental error by any of the above techniques. Thus,transporters that are not expressed in particular tissue exhibit expresslevels less than 0.1% of beta actin, and usually less than 0.01% of betaactin.

Sustained release refers to release of a therapeutic or prophylacticamount of the drug or an active metabolite thereof into the systemicblood circulation over a prolonged period of time relative to thatachieved by oral administration of a conventional formulation of thedrug.

DETAILED DESCRIPTION OF THE INVENTION 1. Introduction

The invention provides methods of screening agents, conjugates orconjugate moieties, linked or linkable to agents, for capacity to betransported as substrates through the PEPT2 transporter. The inventionalso provides methods of treatment involving oral delivery of agentsthat either alone, or as a result of linkage to a conjugate moiety, aresubstrates of the PEPT2 transporter. The present methods are premised,in part, on the inventors' results showing that PEPT2 is expressed inthe human intestine, particularly the stomach, jejunum, ileum, theileo-caecal valve, the cecum and the ascending colon. Previous workershave reported that PEPT2 is present in the brain and kidney but isabsent from the intestine. It is believed that the discrepancy betweenthe present results and previous work may be because most previous workto determine tissues in which PEPT2 is expressed was performed on therat rather than the human, and because of the greater sensitivity ofdetection of quantitative PCR employed in the present examples.

The insight that PEPT2 is expressed in the human intestine opens up newstrategies for design and delivery of drugs through this transporter.Because of the different substrate specificities of PEPT1 and PEPT2,some agents, conjugates or conjugate moieties that are poor substratesfor PEPT1 are transported to a greater extent by PEPT2. Therefore, theavailability of PEPT2 as an alternative transporter to PEPT1 broadensthe range of agents, conjugates and conjugate moieties that can passthrough or facilitate passage through the intestine. Therefore, agents,conjugates or conjugate moieties that are found to be poorly transportedby PEPT1 in screening assays should not necessarily be discarded but canbe retested for transport via PEPT2. Agents, conjugates or conjugatemoieties can also be designed or screened with PEPT2 as the intendedtarget. Expression of PEPT2 in the kidney can result in recirculation ofagents or conjugates for PEPT2 from the kidney back into the systemiccirculation. Reuptake increases the half-life of a drug or conjugatemoiety and hence reduces the dosage that need be administered. Anadvantage of PEPT2 is that its higher affinity for substrates allowstesting of candidate substrates at lower concentrations of the candidatesubstrates than PEPT1. For candidate substrates that are available inonly small amounts or which have low solubilities, the ability todetermine substrate properties at low concentration of substrate is asignificant advantage.

2. PEPT1 and PEPT2

Human PEPT1 has been cloned as a cDNA of 2263 bp with an open readingframe of 2127 bp encoding a protein of 708 amino acids by Liang, Journalof Biological Chemistry 270: 6456-6463 (1995) (SEQ ID NO:1). Referenceto PEPT1 includes the amino acid sequence of Liang, allelic, cognate andinduced variants thereof. Usually such variants show at least 90%sequence identity to the exemplary sequence of Liang. Cognate forms ofthe human PEPT1 sequence have been cloned from rabbit, and rat tissues.Fei, Nature 368: 563-566 (1994), and Miyamoto, Biochimica et BiophysicaActa 1305: 34-38 (1996), respectively.

Human PEPT2 has been cloned by Liu et al., Biochimica et Biophysica Acta1235:461-466 (1995). Reference to PEPT2 includes the amino acid sequenceof Liu et al. (SEQ ID NO:2), allelic cognate and induced variantsthereof. Usually such variants show at least 90% sequence identity tothe exemplary sequence of Liu. Saito et al., Biochimica et BiophysicaActa 1280, 173-177 (1996) have described the isolation of cDNA encodingthe rat H+-coupled peptide transporter PEPT2. The PEPT2 cDNA had 3938bp, which encoded a 729-amino acid protein of a molecular mass of 81kDa. The overall amino acid identity was 48% identical to the rat PEPT1.The rat PEPT2 has twelve putative membrane-spanning α-helices and fourpotential N-linked glycosylation sites at a predicted largeextracellular loop between α-helices 9 and 10. The rat PEPT2 showed 83%amino acid identity to the human PEPT2. The experiments described in theExamples show that human PEPT2 is expressed in the kidney, pancreas,liver, brain, lungs, ileum, jejunum and duodenum among other tissues.PEPT2 is also expressed in the CaCo2 cell line that derives fromintestinal cells.

3. Methods of Identifying Agents, Conjugates or Conjugate Moieties thatare Substrates of the PEPT2 Receptor

Agents known or suspected to have pharmacological activity can bescreened directly for their capacity to act as substrates of the PEPT2transporter. Alternatively, conjugate moieties can be screened assubstrates, and the conjugate moieties linked to agents having known orsuspected pharmacological activity. In such methods, the conjugatemoieties can be linked to an agent or other molecule as a conjugateduring the screening process. If another molecule is used, the moleculeis sometimes chosen to resemble the structure of an agent ultimatelyintended to be linked to the conjugate moiety for pharmaceutical use.The screening is typically performed on cells expressing the PEPT2transporter. In some methods, the cells are transfected with DNAencoding the PEPT2 transporter. In other methods, natural cellsexpressing the PEPT2 transporter are used. In some methods, PEPT2 is theonly transporter or the only peptide transporter expressed. In othermethods, cells express PEPT2 in combination with other transporters. Forexample, in some methods, cells expressing both PEPT1 and PEPT2 areused. In still other methods, agents, conjugates or conjugate moietiesare screened on different cells expressing different transporters. Forexample, agents or conjugates can be screened on cells expressing PEPT2and on cells expressing PEPT1. Methods of screening agents, conjugatesor conjugate moieties for passage through cells bearing a transporterare described in WO 01/20331.

Internalization of a compound evidencing passage through transporterscan be detected by detecting a signal from within a cell from any of avariety of reporters. The reporter can be as simple as a label such as afluorophore, a chromophore, a radioisotope, Confocal imagining can alsobe used to detect internalization of a label as it provides sufficientspatial resolution to distinguish between fluorescence on a cell surfaceand fluorescence within a cell; alternatively, confocal imaging can beused to track the movement of compounds over time. In another approach,internalization of a compound is detected using a reporter that is asubstrate for an enzyme expressed within a cell. Once the complex isinternalized, the substrate is metabolized by the enzyme and generatesan optical signal or radioactive decay that is indicative of uptake.Light emission can be monitored by commercial PMT-based instruments orby CCD-based imaging systems. In addition, assay methods utilizing LCMSdetection of the transported compounds or electrophysiological signalsindicative of transport activity are also employed.

In some methods, multiple agents, conjugates or conjugate moieties arescreened simultaneously and the identity of each agent or conjugatemoiety is tracked using tags linked to the agents, conjugates orconjugate moieties. In some methods, a preliminary step is performed todetermine binding of an agent or conjugate moiety to PEPT2. Although notall agents or conjugates that bind PEPT2 are substrates of thetransporter, observation of binding is an indication that allows one toreduce the number of candidate substrates from an initial repertoire. Insome methods, substrate capacity of an agent or conjugate moiety istested in comparison with a reference substrate of PEPT2. The artificialdipeptide Gly-Sar has often been used as a reference for PEPT1, and canalso be used as a reference for PEPT2. The comparison can either beperformed in separate parallel assays in which an agent or conjugatemoiety under test and Gly-Sar are compared for uptake on separatesamples of the same cells. Alternatively, the comparison can beperformed in a competition format in which an agent or conjugate moietyunder test and Gly-Sar are applied to the same cells. Typically, theagent or conjugate moiety and Gly-Sar are differentially labeled in suchassays.

In such comparative assays, the Vmax of an agent, conjugate moiety, orconjugate comprising an agent and conjugate moiety tested can becompared with that of Gly-Sar. If an agent, conjugate moiety orconjugate has a Vmax of at least 1%, preferably at least 5%, morepreferably at least 10%, even more preferably at least 20%, and mostpreferably at least 50% of Gly-Sar for the PEPT2 transporter then theagent, conjugate moiety or conjugate can be considered to be a substratefor PEPT2. In general, the higher the Vmax of the agent, conjugatemoiety or conjugate relative to that of Gly-Sar the better. Therefore,agents, conjugate moieties or conjugates having Vmax's of at least 50%,100%, 150% or 200% of the Vmax of Gly-Sar for PEPT2 are screened in somemethods. The agents to which conjugate moieties are linked can bythemselves show little or no detectable substrate activity for PEPT2(e.g., Vmax relative to that of Gly-Sar of less than 0.1 or 1%).

In some methods, the Vmax of an agent, conjugate moiety or conjugate isalso determined relative to Gly-Sar for the transporter PEPT1. Suchscreening may reveal that the agent, conjugate moiety or conjugate is abetter substrate for PEPT2 than PEPT1. The relative capacities of asubstrate for PEPT2 and PEPT1 can be compared by a comparison of theratios of Vmax of the agent, conjugate moiety or conjugate and Gly-Sarfor the respective transporters. For example, if the ratio of Vmax's forthe agent, conjugate moiety or conjugate to Gly-Sar is greater for PEPT2than for PEPT1 then the agent, conjugate moiety or conjugate is a bettersubstrate for PEPT2 than for PEPT1. In some methods, the ratio of Vmax'sis at least 2, 10, 20, 50, or 100 times greater for PEPT2 than forPEPT1. In some methods, the ratio of the agent, conjugate moiety orconjugate to Gly-Sar for PEPT1 is less than 0.1, 1 or 10%. In othermethods, the agent, conjugate moiety or conjugate is a substrate forPEPT1 (Vmax of at least 10%, 50%, 100%, 150% or 200% of the Vmax ofGly-Sar for PEPT1. Robust assays are available for both PEPT1 and PEPT2,allowing design and characterization of compounds with substrate (orinhibitor) activities for either PEPT1 or PEPT2, or both. Based on theconventional wisdom, compounds lacking substrate activity on PEPT1 wouldbe rejected as candidates for oral delivery; however, based on ourdetection of significant PEPT2 expression in human intestine, compoundstransported by PEPT2 can be recognized and optimized for oral deliverythrough PEPT2 transporters in the human intestine).

4. Agents, Conjugates and Conjugate Moieties to be Screened

Compounds constituting agents, conjugates or conjugate moieties to bescreened can be naturally occurring or synthetic molecules. Naturalsources include sources such as, e.g., marine microorganisms, algae,plants, and fungi. Alternatively, compounds to be screened can be fromcombinatorial libraries of agents, including peptides or smallmolecules, or from existing repertories of chemical compoundssynthesized in industry, e.g., by the chemical, pharmaceutical,environmental, agricultural, marine, cosmeceutical, drug, andbiotechnological industries. Compounds can include, e.g.,pharmaceuticals, therapeutics, environmental, agricultural, orindustrial agents, pollutants, cosmeceuticals, drugs, organic compounds,lipids, glucocorticoids, antibiotics, peptides, sugars, carbohydrates,and chimeric molecules.

A variety of methods are available for producing peptide libraries (see,e.g., Lam et al., Nature, 354: 82, 1991 and WO 92/00091; Geysen et al.,J Immunol Meth, 102: 259, 1987: Houghten et al., Nature, 354: 84, 1991and WO 92/09300 and Lebl et al., Int J Pept Prot Res, 41, 201, 1993).Peptide libraries can also be generated by phage display methods. See,e.g., Dower, U.S. Pat. No. 5,723,286.

Combinatorial libraries can be produced for many types of compounds thatcan be synthesized in a step-by-step fashion (see e.g., Ellman & Bunin,J Amer Chem Soc, 114:10997, 1992 (benzodiazepine template), WO 95/32184(oxazolone and aminidine template), WO 95/30642 (dihydrobenzopyrantemplate) and WO 95/35278 (pyrrolidine template). Libraries of compoundsare usually synthesized by solid phase chemistry on particle. However,solution-phase library synthesis can also be useful. Strategies forcombinatorial synthesis are described by Dol+ Le & Nelson, J.Combinatorial Chemistry 1. 235-282 (1999)) (incorporated by reference inits entirety for all purposes). Synthesis is typically performed in acyclic fashion with a different monomer or other component being addedin each round of synthesis. Some methods are performed by successivelyfractionating an initial pool. For example, a first round of synthesisis performed on all supports. The supports are then divided into twopools and separate synthesis reactions are performed on each pool. Thetwo pools are then further divided, each into a further two pools and soforth. Other methods employ both splitting and repooling. For example,after an initial round of synthesis, a pool of compounds is split intotwo for separate syntheses in a second round. Thereafter, aliquots fromthe separate pools are recombined for a third round of synthesis. Splitand pool methods result in a pool of mixed compounds. These methods areparticularly amenable for tagging as described in more detail below. Thesize of libraries generated by such methods can vary from 2 differentcompounds to 10⁴, 10⁶, 10⁸, or 10¹⁰, or any range therebetween.

Preparation of encoded libraries is described in a variety ofpublications including Needels, et al., Proc. Natl. Acad. Sci. USA 1993,90, 10700; Ni, et al., J. Med. Chem. 1996, 39, 1601, WO 95/12608, WO93/06121, WO 94/08051, WO 95/35503 and WO 95/30642 (each of which isincorporated by reference in its entirety for all purposes). Methods forsynthesizing encoded libraries typically involve a random combinatorialapproach and the chemical and/or enzymatic assembly of monomer units.For example, the method typically includes steps of: (a) apportioning aplurality of solid supports among a plurality of reaction vessels; (b)coupling to the supports in each reaction vessel a first monomer and afirst tag using different first monomer and tag combinations in eachdifferent reaction vessel; (c) pooling the supports; (d) apportioningthe supports among a plurality of reaction vessels; (e) coupling to thefirst monomer a second monomer and coupling to either the solid supportor to the first tag a second tag using different second monomer andsecond tag combinations in each different reaction vessel; andoptionally repeating the coupling and apportioning steps with differenttags and different monomers one to twenty or more times. The monomer setcan be expanded or contracted from step to step; or the monomer setcould be changed completely for the next step (e.g., amino acids in onestep, nucleosides in another step, carbohydrates in another step). Amonomer unit for peptide synthesis, for example, can include singleamino acids or larger peptide units, or both.

Compounds synthesizable by such methods include polypeptides, beta-turnmimetics, polysaccharides, phospholipids, hormones, prostaglandins,steroids, aromatic compounds, heterocyclic compounds, benzodiazepines,oligomeric N-substituted glycines and oligocarbamates. Preparedcombinatorial libraries are also available from commercial sources(e.g., ChemRx, South San Francisco, Calif.).

Some compounds to be screened are variants of known transportersubstrates. The natural function of these transporters is to transportpeptides arising from the digestion of dietary proteins (smallintestine) and prevent loss of peptides in the glomerular filtrate(kidney). Some compounds to be screened are peptides, variants of aminoacids, zwitterionic antibiotics, sugars or nucleosides, or structuralvariants of any of these. Compounds to be screened also include variantsof known substrates, such as β-lactam antibiotics, the anti-cancer agentBestatin, and angiotensin converting enzyme (ACE) inhibitors. Orallybioavailable antibiotics interact differently with PEPT1 and PEPT2. Ingeneral, although not invariably, β-lactam antibiotics having an α-aminogroup (cefadroxil, cephradine, amoxacillin, and cyclacillin) are bettersubstrates for PEPT2 than PEPT1. β-lactam antibiotics without α-aminogroups (ceftibuten, cefixime, and cefdinir) are not as good substratesfor PEPT2, but are moderate substrates for PEPT1.

5. Linkage of Agents to Conjugate Moieties

Conjugate moieties that are substrates for PEPT2 or other transportercan be attached to or incorporated into agents having pharmacologicalactivity by a variety of means. Conjugates of this invention can beprepared by either direct conjugation of an agent to a conjugate moiety,wherein the resulting covalent bond is cleavable in vivo, or bycovalently coupling a difunctionalized linker precursor with an agent toa conjugate moiety. The linker precursor is selected to contain at leastone reactive functionality that is complementary to at least onereactive functionality on the agent and at least one reactivefunctionality on the conjugate moiety. Such complementary reactivegroups are well known in the art as illustrated below:

COMPLEMENTARY BINDING CHEMISTRIES First Reactive Second Reactive GroupGroup Linkage hydroxyl carboxylic acid ester hydroxyl haloformatecarbonate thiol carboxylic acid thioester thiol haloformatethiocarbonate amine carboxylic acid amide hydroxyl isocyanate carbamatehydroxyl haloformate carbamate amine isocyanate urea carboxylic acidcarboxylic acid anhydride hydroxyl phosphorus acid phosphonate orphosphate ester

In addition to the complementary chemistry of the functional groups onthe linker to both the agent and conjugate moiety, the linker (whenemployed) is also selected to be cleavable in vivo. Cleavable linkersare well known in the art and are selected such that at least one of thecovalent bonds of the linker that attaches the agent to the conjugatemoiety can be broken in vivo thereby providing for the agent or activemetabolite thereof to be available to the systemic blood circulation.The linker is selected such that the reactions required to break thecleavable covalent bond are favored at the physiological site in vivowhich permits agent (or active metabolite thereof) release into thesystemic blood circulation. The selection of suitable cleavable linkersto provide effective concentrations of the agent or active metabolitethereof for release into the systemic blood circulation can be evaluatedusing endogenous enzymes in standard in vitro assays to provide acorrelation to in vivo cleavage of the agent or active metabolitethereof from the conjugate, as is well known in the art. It isrecognized that the exact cleavage mechanism employed is not critical tothe methods of this invention provided, of course, that the conjugatecleaves in vivo in some form to provide for the agent or activemetabolite thereof for sustained release into the systemic bloodcirculation.

In another approach, a conjugate moiety and agent are each attached tomoieties having mutual affinity for each (e.g., avidin or streptavidinand biotin, or hexahistidine and Ni²⁺). In another approach, both agentand conjugate moiety are linked to a solid phase. Examples of suchsupports include nanoparticles (see, e.g., U.S. Pat. Nos. 5,578,325 and5,543,158), molecular scaffolds, liposomes (see, e.g., Deshmuck, D. S.,et al., Life Sci. 28:239-242 (1990), and Aramaki, Y., et al., Pharm.Res. 10:1228-1231 (1993), protein cochleates (stableprotein-phospholipid-calcium precipitates; see, e.g., Chen et al., J.Contr. Rel. 42:263-272 (1996), and clathrate complexes. These supportscan be used to attach other active molecules. Certain supports such asnanoparticles can also be used to encapsulate desired compounds. Anagent can be linked to a support via a cleavable linkage allowingseparation of the agent after uptake through a transporter.

Examples of cleavable linkers suitable for use as described aboveinclude nucleic acids with one or more restriction sites, or peptideswith protease cleavage sites (see, e.g., U.S. Pat. No. 5,382,513). Otherexemplary linkers that can be used are available from Pierce ChemicalCompany in Rockford, Ill.; suitable linkers are also described in EPA188,256; U.S. Pat. Nos. 4,671,958; 4,659,839; 4,414,148; 4,699,784;4,680,338, 4,569,789 and 4,590,071; and in Eggenweiler, H. M, DrugDiscovery Today, 3: 552 (1998), each of which is incorporated in itsentirety for all purposes.

There are many existing drugs for which uptake can be improved throughthe intestine. Drugs suitable for conversion to prodrugs that arecapable of uptake from the intestine typically contain one or more ofthe following functional groups to which a promoiety may be conjugated:primary or secondary amino groups, hydroxyl groups, carboxylic acidgroups, phosphonic acid groups, or phosphoric acid groups.

Examples of drugs containing carboxyl groups include, for instance,angiotensin-converting enzyme inhibitors such as alecapril, captopril,1-[4-carboxy-2-methyl-2R,4R-pentanoyl]-2,3-dihydro-2S-indole-2-carboxylicacid, enalaprilic acid, lisinopril,N-cyclopentyl-N-[3-[(2,2-dimethyl-1-oxopropyl)thio]-2-methyl-1-oxopropyl]glycine,pivopril, quinaprilat,(2R,4R)-2-hydroxyphenyl)-3-(3-mercaptopropionyl)-4-thiazolidinecarboxylicacid, (S) benzamido-4-oxo-6-phenylhexenoyl-2-carboxypyrrolidine,[2S-1[R*(R*))]]2α, 3αβ,7αβ]-1[2-[[1-carboxy-3-phenylpropyl]-amino]-1-oxopropyl]octahydro-1H-indole-2-carboxylicacid, [3S-1[R*(R*))]],3R*]-2-[2-[[1-carboxy-3-phenylpropyl]-amino]-1-oxopropyl]-1,2,3,4-tetrahydro-3-isoquinolonecarboxylic acid, and tiopronin; cephalosporin antibiotics such ascefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazuflur,cefazolin, cefbuperazone, cefixime, cefinenoxime, cefinetazole,cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotefan,cefotiam, cefoxitin, cefpimizole, cefpirome, cefpodoxime, cefroxadine,cefsulodin, cefpiramide, ceftazidime, ceftezole, ceftizoxime,ceftriaxone, cefuroxime, cephacetrile, cephalexin, cephaloglycin,cephaloridine, cephalosporin, cephanone, cephradine, and latamoxef;penicillins such as amoxycillin, ampicillin, apalcillin, azidocillin,azlocillin, benzylpenicillin, carbenicillin, carfecillin, carindacillin,cloxacillin, cyclacillin, dicloxacillin, epicillin, flucloxacillin,hetacillin, methicillin, mezlocillin, nafcillin, oxacillin,phenethicillin, piperazillin, sulbenicllin, temocillin, and ticarcillin;thrombin inhibitors such as argatroban, melagatran, and napsagatran;influenza neuraminidase inhibitors such as zanamivir and peramivir;non-steroidal antiinflammatory agents such as acametacin, alclofenac,alminoprofen, aspirin (acetylsalicylic acid), 4-biphenylacetic acid,bucloxic acid, carprofen, cinchofen, cinmetacin, clometacin, clonixin,diclenofac, diflunisal, etodolac, fenbufen, fenclofenac, fenclosic acid,fenoprofen, ferobufen, flufenamic acid, flufenisal, flurbiprofin,fluprofen, flutiazin, ibufenac, ibuprofen, indomethacin, indoprofen,ketoprofen, ketorolac, lonazolac, loxoprofen, meclofenamic acid,mefenamic acid,2-(8-methyl-10,11-dihydro-11-oxodibenz[b,f]oxepin-2-yl)propionic acid,naproxen, nifluminic acid, O-(carbamoylphenoxy)acetic acid, oxoprozin,pirprofen, prodolic acid, salicylic acid, salicylsalicylic acid,sulindac, suprofen, tiaprofenic acid, tolfenamic acid, tolmetin andzopemirac; prostaglandins such as ciprostene,16-deoxy-16-hydroxy-16-vinyl prostaglandin E₂,6,16-dimethylprostaglandin E₂, epoprostostenol, meteneprost, nileprost,prostacyclin, prostaglandins E₁, E₂, or F_(2α), and thromboxane A₂;quinolone antibiotics such as acrosoxacin, cinoxacin, ciprofloxacin,enoxacin, flumequine, naladixic acid, norfloxacin, ofloxacin, oxolinicacid, pefloxacin, pipemidic acid, and piromidic acid; other antibioticssuch as aztreonam, imipenem, meropenem, and related carbopenemantibiotics.

Representative drugs containing amine groups include: acebutalol,albuterol, alprenolol, atenolol, bunolol, bupropion, butopamine,butoxamine, carbuterol, cartelolol, colterol, deterenol, dexpropanolol,diacetolol, dobutamine, exaprolol, exprenolol, fenoterol, fenyripol,labotolol, levobunolol, metolol, metaproterenol, metoprolol, nadolol,pamatolol, penbutalol, pindolol, pirbuterol, practolol, prenalterol,primidolol, prizidilol, procaterol, propanolol, quinterenol, rimiterol,ritodrine, solotol, soterenol, sulfiniolol, sulfinterol, sulictidil,tazaolol, terbutaline, timolol, tiprenolol, tipridil, tolamolol,thiabendazole, albendazole, albutoin, alendronate, alinidine,alizapride, amiloride, aminorex, aprinocid, cambendazole, cimetidine,cisapride, clonidine, cyclobenzadole, delavirdine, efegatrin,etintidine, fenbendazole, fenmetazole, flubendazole, fludorex,gabapentin, icadronate, lobendazole, mebendazole, metazoline,metoclopramide, methylphenidate, mexiletine, neridronate, nocodazole,oxfendazole, oxibendazole, oxmetidine, pamidronate, parbendazole,pramipexole, prazosin, pregabalin, procainamide, ranitidine,tetrahydrazoline, tiamenidine, tinazoline, tiotidine, tocainide,tolazoline, tramazoline, xylometazoline, dimethoxyphenethylamine,N-[3(R)-[2-piperidin-4-yl)ethyl]-2-piperidone-1-yl]acetyl-3(R)-methyl-β-alanine,adrenolone, aletamine, amidephrine, amphetamine, aspartame, bamethan,betahistine, carbidopa, clorprenaline, chlortermine, dopamine, L-Dopa,ephrinephrine etryptamine, fenfluramine, methyldopamine, norepinephrine,tocainide, enviroxime, nifedipine, nimodipine, triamterene, norfloxacin,and similar compounds such as pipedemic acid,1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-1,8-napthyridine-3-carboxylicacid, 1-cyclopropyl-6-fluoro-1, and4-dihydro-4-oxo-7-(piperazinyl)-3-quinolinecarboxylic acid.

Representative drugs containing hydroxy groups include: steroidalhormones such as allylestrenol, cingestol, dehydroepiandrosteron,dienostrol, diethylstilbestrol, dimethisteron, ethyneron, ethynodiol,estradiol, estron, ethinyl estradiol, ethisteron, lynestrenol,mestranol, methyl testosterone, norethindron, norgestrel, norvinsteron,oxogeston, quinestrol, testosterone, and tigestol; tranquilizers such asdofexazepam, hydroxyzin, lorazepam, and oxazepam; neuroleptics such asacetophenazine, carphenazine, fluphenazine, perphenyzine, andpiperaetazine; cytostatics such as aclarubicin, cytarabine, decitabine,daunorubicin, dihydro-5-azacytidine, doxorubicin, epirubicin,estramustin, etoposide, fludarabine, gemcitabine,7-hydroxychlorpromazin, nelarabine, neplanocin A, pentostatin,podophyllotoxin, tezacitabine, troxacitabine, vinblastin, vincristin,and vindesin; hormones and hormone antagonists such as buserilin,gonadoliberin, icatibrant, and leuprorelin acetate; antihistamines suchas terphenadine; analgesics such as diflunisal, naproxol, paracetamol,salicylamide, and salicyclic acid; antibiotics such as azidamphenicol,azithromycin, camptothecin, cefamandol, chloramphenicol, clarithromycin,clavulanic acid, clindamycin, demeclocyclin, doxycyclin, erythromycin,gentamycin, imipenem, latamoxef, metronidazole, neomycin, novobiocin,oleandomycin, oxytetracyclin, tetracycline, thiamenicol, and tobramycin;antivirals such as acyclovir, dideoxydidehydrocytidine, dideoxycytosine,1-(2-deoxy-2-methylene-beta-D-erythro-pentofuranosyl)cytidine,fluoro-dideoxydidehydrocytidine, fluorodideoxycytosine, FMAU(1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine),deoxy-5-fluoro-3′-thiacytidine, 2′-fluoro-ara-dideoxyinosine,ganciclovir, lamivudine, penciclovir, SddC, stavudine,5-trifluoromethyl-2′-deoxyuridine, zalcitabine, and zidovudine;bisphosphonates such as EB-1053(1-hydroxy-3-(1-pyrrolidinyl)propylidene-1,1-bisphosphonate),etidronate, ibandronate, olpadronate, residronate,1-hydroxy-2-(imidazo[1,2-a]pyridin-3-yl)ethylidene]-bisphosphonic acid,and zolendronate; protease inhibitors such as ciprokiren, enalkiren,ritonavir, saquinavir, and terlakiren; prostaglandins such asarbaprostil, carboprost, misoprostil, and prostacydin; antidepressivessuch as 8-hydroxychlorimipramine and 2-hydroxyimipramine;antihypertonics such as sotarol and fenoldopam; anticholinerogenics suchas piperidine, procyclidin and trihexyphenidal; antiallergenics such ascromolyn; glucocorticoids such as betamethasone, budenosid,chlorprednison, clobetasol, clobetasone, corticosteron, cortisone,cortodexon, dexamethason, flucortolon, fludrocortisone, flumethasone,flunisolid, fluprednisolon, flurandrenolide, flurandrenolon acetonide,hydrocortisone, meprednisone, methylpresnisolon, paramethasone,prednisolon, prednisol, triamcinolon, and triamcinolon acetonide;narcotic agonists and antagonists such as apomorphine, buprenorphine,butorphanol, codein, cyclazocin, hydromorphon, ketobemidon,levallorphan, levorphanol, metazocin, morphine, nalbuphin, nalmefen,naloxon, nalorphine, naltrexon, oxycodon, oxymorphon, and pentazocin;stimulants such asmazindol and pseudoephidrine; anaesthetics such ashydroxydion and propofol; β-receptor blockers such as acebutolol,albuterol, alprenolol, atenolol, betazolol, bucindolol, cartelolol,celiprolol, cetamolol, labetalol, levobunelol, metoprolol, metipranolol,nadolol, oxyprenolol, pindolol, propanolol, and timolol;α-sympathomimetics such as adrenalin, metaraminol, midodrin,norfenefrin, octapamine, oxedrin, oxilofrin, oximetazolin, andphenylefrin; β-sympathomimetics such as bamethan, clenbuterol,fenoterol, hexoprenalin, isoprenalin, isoxsuprin, orciprenalin,reproterol, salbutamol, and terbutalin; bronchodilators such ascarbuterol, dyphillin, etophyllin, fenoterol, pirbuterol, rimiterol andterbutalin; cardiotonics such as digitoxin, dobutamin, etilefrin, andprenalterol; antimycotics such as amphotericin B, chlorphenesin,nystatin, and perimycin; anticoagulants such as acenocoumarol,dicoumarol, phenprocoumon, and warfarin; vasodilators such as bamethan,dipyrimadol, diprophyllin, isoxsuprin, vincamin and xantinol nicotinate;antihypocholesteremics such as compactin, eptastatin, mevinolin, andsimvastatin; miscellaneous drugs such as bromperidol (antipsychotic),dithranol (psoriasis) ergotamine (migraine) ivermectin (antihelminthic),metronidazole and secnizadole (antiprotozoals), nandrolon (anabolic),propafenon and quinadine (antiarythmics), quetiapine (CNS), serotonin(neurotransmitter), and silybin (hepatic disturbance).

Representative drugs containing phosphonic acid moieties include:adefovir, alendronate,(N6-[2-methylthio)ethyl]-2-[3,3,3-trifluoropropylthio]-5′-adenylic acid,BMS-187745 (a squalene synthase inhibitor from Bristol-Meyers SquibbInc.), ceronapril, CGP-24592 (Novartis, Inc.),DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid; 4-methyl-APPA,CGP-39551 (ethyl esters of(DL-[E]-2-amino-4-methyl-5-phosphono-3-pentenoic acid)), CGP-40116 (acompetitive NMDA antagonist by Novartis Inc.), cidofovir, clodronate,EB-1053 (1-hydroxy-3-(1-pyrrolidinyl)propylidene-1,1-bisphosphonate),etidronate, fanapanel, foscarnet, fosfomycin, fosinopril, fosinoprilat,ibandronate, midafotel, neridronate, olpadronate, pamidronate,residronate, tenofovir, tiludronate,[2-(8,9-dioxo-2,6-diazabicyclo[5.2.0]non-1(7)-en-2-yl)ethyl]phosphonicacid, 1-hydroxy-2-(imidazo[1,2-a]pyridin-3-yl)ethylidene]-bisphosphonicacid, and zolendronate.

Representative drugs containing phosphoric acid moieties include:bucladesine, choline alfoscerate, citocoline, fludarabine phosphate,fosopamine, GP-668, perifosine, triciribine phosphate, and phosphatederivatives of nucleoside analogs which require phosphorylation foractivity, such as lamivudine, acyclovir, azidothymidine,E-5-(2-bromovinyl)-2′-deoxyuridine, dideoxycytosine, dideoxyinosine,FMAU (1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine),deoxy-5-fluoro-3′-thiacytidine, ganciclovir, gemcitabine,(R)-9-[4-Hydroxy-2-(hydroxymethy)butyl]guanine, lamivudine, penciclovirand the like.

Preferred drugs for modification to prodrugs capable of intestionalabsorption and incorporation into sustained release formulations includethe following compounds: analgesics and/or antiinflammatory agentsselected from the group consisting of acetaminophen, buprenorphine,diclofenac, diflunisal, fenoprofen, ibuprofen, indomethacin, ketoprofen,mefenamic acid, meptazinol, morphine, oxycodone, pentazocine, pethidine,tolmetin, and tramadol; antihypertensive agents selected from the groupconsisting of captopril, diltiazem, methyldopa, metoprolol, prazosin,propranolol, quinapril, sotalol, and timolol; antibiotic agents selectedfrom the group consisting of amoxicillin, ampicillin, aztreonam,cefaclor, cefadroxil, cefixime, cefotaxime, cefoxitin, cefpodoxime,ceftizoxime, ceftriaxone, cefuroxime, cephalexin, ciproflaxacin,clindamycin, erythromycin, imipenem, mandol, meropenem, metronidazole,and tobramycin; antiviral agents selected from the group consisting ofacyclovir, delavirdine, didanosine, foscarnet, ganciclovir, indinavir,lamivudine, nelfinavir, penciclovir, ritonavir, saquinavir, stavudine,zalcitabine, and zidovudine; bronchodilator and or anti-asthmatic agentsselected from the group consisting of salbutamol and terbutaline;antiarrhythmic agents selected from the group consisting of mexiletine,procainamide, and tocainide; centrally acting substances selected fromthe group consisting of baclofen, benserazide, bupropion, carbidopa,gabapentin, levodopa, methylphenildate, pramipexole, pregabalin,quetiapine, ropinirole, and vigabatrin; cytostatics and metastasisinhibitors selected from the group consisting of cytarabine, decitabine,docetaxal, flutamide, gemcitabine, paclitaxel, and pentostatin; and,agents for treatment of gastrointestinal disorders selected from thegroup consisting of cisapride, metoclopramide, and misoprostol.

6. Pharmaceutical Compositions and Methods of Treatment

Agents that are themselves substrates for PEPT2 or which are linked toconjugate moieties that are substrates for PEPT2 can be can beincorporated into pharmaceutical compositions. Usually, although notnecessarily, such pharmaceutical compositions are designed for oraladministration. Oral administration of such compositions results inuptake through the intestine via the PEPT2 and entry into the systemiccirculation. The pharmaceutical composition can thus be efficientlydelivered to a wide range of tissues in the body. The specificity ofcompositions for PEPT2 renders the compositions susceptible to uptake bythe brain (including the choroid plexus) and kidney that express PEPT2at high levels. However, the methods are also useful for treating a widevariety of diseases in patients who are free of diseases of the brain,kidney, lung, and spleen in which PEPT2 is expressed to a significantextent. In such methods, the expression of PEPT2 in the kidney increasereabsorption of the pharmaceutical composition into the systemiccirculation thereby increasing its half life and thereby reducing thedosage necessary. In some methods, the agent or conjugate moiety is asubstrate for both PEPT2 and PEPT1. In some methods, the agent orconjugate moiety is a substrate for PEPT2 and is not a substrate, or isa poor substrate, for PEPT1.

Agents optionally linked to a conjugate moiety are combined withpharmaceutically-acceptable, non-toxic carriers of diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to adversly affect the biological activity of the combination.Examples of such diluents are distilled water, buffered water,physiological saline, PBS, Ringer's solution, dextrose solution, andHank's solution. In addition, the pharmaceutical composition orformulation can also include other carriers, adjuvants, or non-toxic,nontherapeutic, nonimmunogenic stabilizers, excipients and the like. Thecompositions can also include additional substances to approximatephysiological conditions, such as pH adjusting and buffering agents,toxicity adjusting agents, wetting agents, detergents and the like (see,e.g., “Remington's Pharmaceutical Sciences”, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985); for a brief review of methods fordrug delivery, see, Langer, Science 249:1527-1533 (1990); each of thesereferences is incorporated by reference in its entirety).

Pharmaceutical compositions for oral administration can be in the formof e.g., tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, or syrups. Some examples of suitableexcipients include lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, sterile water, syrup, and methyl cellulose. Preserving agentssuch as methyl- and propylhydroxy-benzoates; sweetening agents; andflavoring agents can also be included. Depending on the formulation,compositions can provide quick, sustained or delayed release of theactive ingredient after administration to the patient. The tablets orpills of the present invention may be coated or otherwise compounded toprovide a dosage form affording the advantage of prolonged action. Forexample, the tablet or pill can comprise an inner dosage and an outerdosage component, the latter being in the form of an envelope over theformer. The two components can be separated by an enteric layer whichserves to resist disintegration in the stomach and permit the innercomponent to pass intact into the duodenum or to be delayed in release.A variety of materials can be used for such enteric layers or coatings,such materials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 mg to about 2 g of the active agent.

The compositions can be administered for prophylactic and/or therapeutictreatments. A therapeutic amount is an amount sufficient to remedy adisease state or symptoms, or otherwise prevent, hinder, retard, orreverse the progression of disease or any other undesirable symptoms inany way whatsoever. In prophylactic applications, compositions areadministered to a patient susceptible to or otherwise at risk of aparticular disease or infection. Hence, a “prophylactically effectiveamount” is an amount sufficient to prevent, hinder or retard a diseasestate or its symptoms. In either instance, the precise amount ofcompound contained in the composition depends on the patient's state ofhealth and weight.

An appropriate dosage of the pharmaceutical composition is readilydetermined according to any one of several well-established protocols.For example, animal studies (e.g., mice, rats) are commonly used todetermine the maximal tolerable dose of the bioactive agent per kilogramof weight. In general, at least one of the animal species tested ismammalian. The results from the animal studies can be extrapolated todetermine doses for use in other species, such as humans for example.

The pharmaceutical compositions can be administered in a variety ofdifferent ways. Examples include administering a composition containinga pharmaceutically acceptable carrier via oral, intranasal, rectal,topical, intraperitoneal, intravenous, intramuscular, subcutaneous,subdermal, transdermal, intrathecal, and intracranial methods. The routeof administration depends in part on the chemical composition of theactive compound and any carriers.

The components of pharmaceutical compositions are preferably of highpurity and are substantially free of potentially harmful contaminants(e.g., at least National Food (NF) grade, generally at least analyticalgrade, and more typically at least pharmaceutical grade). To the extentthat a given compound must be synthesized prior to use, the resultingproduct is typically substantially free of any potentially toxic agents,particularly any endotoxins, which may be present during the synthesisor purification process. Compositions for parental administration arealso sterile, substantially isotonic and made under GMP conditions.Compositions for oral administration need not be sterile orsubstantially isotonic but are usually made under GMP conditions.

EXAMPLES 1. PCR Analysis of Transporter Expression

Oligonucleotide primers were designed to amplify specific sequences ineither human PEPT1 (2 sets using Genbank) or PEPT2 (2 sets usingGenbank). The forward and reverse primer sequences were (PEPT1#1F-catgcaccaccacgcccagctatttt (SEQ ID NO:3) andR-gcgcggtagctcaagcctgtaatccc (SEQ ID NO:4) which amplifies 147 basepairs in the 3′UTR, PEPT1#2 F-ccgcgttgcttctggtcgtctgtgta (SEQ ID NO:5)and R-tccatcctccacttgcctcctgacct (SEQ ID NO:6) which amplifies 197 basepairs across the stop codon; PEPT2#1 F-acaaccaatgggatgacaaccgtgag (SEQID NO:7) and R-aggcagatcaccagcaggaggcagga (SEQ ID NO:8) which amplifies533 base pairs in the PEPT2 open reading frame; PEPT2#2F-caatgttggtgaagactatggtgtgt (SEQ ID NO:9) andR-aacaagcacgatgatattcccaactg (SEQ ID NO:10) which amplifies the last 376base pairs in the PEPT2 open reading frame). All primers had annealingtemperatures above 55° C. and products were sequenced to verifyspecificity.

Transporter expression was quantitated by PCR (polymerase chainreaction) amplification using real-time PCR (Cepheid Smartcycler PCRinstrument and Perkin-Elmer SYBR-green reagents; all protocols permanufacturers specifications). Single-stranded cDNA was prepared fromhuman mRNA (purchased from Clontech, BioChain, and Stratagene) ordifferentiated Caco-2 cells (Qiagen RNA purification columns) usingThermoscript (Stratagene) reverse transcriptase kit. Real-time PCR wasperformed using the primer sets listed above to amplify fragments ofhuman PEPT1 or PEPT2. In addition, total mRNA abundance was normalizedby measurement of β-Actin levels in each tissue (Clontech primer set).Transcript abundance was measured by determining the threshold cycle forPEPT1 or PEPT2 and calculating transcript number using a calibrationfactor derived from amplification of known plasmid copy numbers. Tocompare different tissues, all data are expressed as a fraction ofβ-actin transcript levels.

Table 1 shows expression levels of PEPT1 and PEPT2 mRNA expressed as apercentage of the expression level of beta actin mRNA in the sametissue. It can be seen that substantial expression of PEPT2 is obtainedin the human jejunum, ileum, ileocecum, and cecum and detectableexpression in several other intestinal tissues. Levels of expression inthe rat duodenum, jejunum and ileum and colon were barely detectable.

TABLE 1 Expression of PEPT1 and PEPT2 in Various Tissues and Cell linesD. Sto Eso Duo Jej Ile Il-Ce Cec ACol TCol Col Hea Bra Lun SMus Kid PanPEPT1 U13173 18.96 2.97 4.92 4.52 3.74 1.13 0.28 0.31 5.27 12.12 5.278.25 0.00 16.68 5.62 7.26 PEPT2 NM_021082 0.05 0.01 0.10 0.77 0.52 0.290.11 0.07 0.01 0.01 0.01 0.09 0.08 0.01 1.14 0.46 HCT8 HT29 Liv Thy SplLeu Pla Pros Test Ova Caco-2 Diff. Undiff. Diff. Undiff. Diff. PEPT1U13173 4.08 12.12 21.54 24.48 12.12 0.00 0.00 10.00 3.31 30.24 16.633.06 2.78 9.18 77.43 PEPT2 NM_021082 0.00 0.03 0.09 0.02 0.01 0.53 0.110.00 0.09 0.76 0.26 0.01 0.18 0.01 0.64 GB# = GenBank accession numberSto = stomach Eso = esophogus Duo = duodenum Jej = jejunum Ile = ileumIl-Ce = ileum-cecum valve Cec = cecum Acol = ascending colon Tcol =total colon Dcol = descending colon Hea = heart Bra = Brain Lun = lungSMus = smooth muscle Kid = kidney Pan = pancreas Liv = liver thy =thymus spl = spleen Leu = leukocytes Pla = platelets Pros = prostatetest = testes Ova = ovaries

2. Functional Analysis of PEPT1 and PEPT2

The complete open reading frame was cloned into a Xenopus oocyteexpression plasmid, linearized, and cRNA was generated by run-offtranscription using the T7 polymerase. Xenopus oocytes were prepared andmaintained as previously described

(Collins, et al., 1997) and injected with 10-30 ng RNA. Transportcurrents were measured 2-4 days after injection using two-electrodevoltage-clamp (Axon Instruments). All experiments were performed using amodified oocyte Ringers solution (90 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1mM MgCl2, and 10 mM NaHEPES, pH 6.8). The membrane potential of oocyteswas held at −60 mV and current traces acquired using PowerLab software.Responses to compounds were measured in the presence and absence of aspecific non-transported inhibitor (XP10973) for PEPT1 and PEPT2. Dataare expressed as the currents that are blocked by XP10973

Stable clones of CHOK1 cells were obtained by electroporation, selectionin G418, and sorting into single-clones using flow-activated cellsorting (Cytomation). Stable clones expressing PEPT1 or PEPT2 wereidentified by enhanced uptake of radiolabeled Gly-Sar. For cell uptakestudies, CHOK1 clones were seeded into polylysine coated 96-wellmicrotitre plates and grown for 2-3 days. Cells were incubated withexperimental solutions (combinations of radiolabeled and unlabeledcompounds) for 30 minutes, washed four times, and either lysed inscintillation solution or water. Uptake of unlabeled compounds wasquantitated by LC/MS/MS.

Table 2 shows the Vmax of several commercial compounds in oocytestransfected with PEPT1 or PEPT2 compared with Vmax of control Gly-Sar.The table also shows Vmax in the presence of the inhibitor XP10973.

XP10973 is a specific inhibitor of both PEPT1 and PEPT2. It can be seenthat cephradine, cephydroxil, cefaclor and amoxacillin are relativelypoor susbstrates relative to Gly-Sar for cells transfected with PEPT1.However, these compounds have Vmax's comparable to or greater than thatof Gly-Sar for cells transfected with PEPT2 indicating that thecompounds are relatively good substrates for PEPT2. This conclusion isreinforced by the data in the presence of the XP10973 inhibitor. Theresult that XP10973 inhibits transport in both PEPT1 and PEPT2transfected cells indicates that transport in such cells is at least inpart due to the PEPT1 and PEPT2 transporters respectively. The smallerextent of inhibition for most substrates for PEPT1 relative to PEPT2indicates that nonspecific transport mechanisms make a more significantrelative contribution in the cells transfected with PEPT1. Except forcephydroxil, treatment with XP1097 results in a lesser percentagedecrease in Vmax for oocytes transfected with PEPT1 relative to oocytestransfected with PEPT2. In short, the experiment shows that cefaclor,cefadroxil and cephradine are better substrates for PEPT2 than they arefor PEPT1. Because it is known that the commercial compounds are orallyavailable, it is probable that they are taken up through the mechanismby an alternate transporter, such as PEPT2.

TABLE 2 PEPT1 (% Gly-Sar) PEPT2 (% Gly-Sar) Drug drug only +XP10973 drugonly +XP10973 Gly-Sar 100 16 100 12 Cephradine 3.5 1.9 159 37Cephadroxil 18 2.1 96 27 Cefaclor 4 1.6 148 22 Amoxacillin 1.3 1.1 85 15

3. Analysis of Uptake in Differentiated Caco-2 Cells

Caco-2 cells were plated on Millipore transwell filters and allowed todifferentiate for 18-22 days. Integrity of the monolayers was confirmedby lack of radiolabeled inulin transport across the monolayer. Compoundswere added to the apical chamber, and the appearance of compounds in thebasolateral chamber were measured at various timepoints by scintillationcounting or LC/MS/MS.

FIGS. 1 and 2 show uptake of cephradine and cefadroxil by Caco-2 cellsin the presence and absence of XP10973 inhibitor. As shown in Table 1,Caco-2 cells express both the PEPT1 and PEPT2 transporters. However, asdescribed above, cephradine and cefadroxil are poor substrates for PEPT1and good substrates for PEPT2. The figures show that both cephradine andcefadroxil are taken up by the Caco-2 cells and that uptake is inhibitedby XP10973. It can be inferred from these results that the cephradineand cefadroxil are taken up via the PEPT2 transporter.

4. Procedure for Preparing a Library to Explore for PEPT2 SpecificSubstrates

Into twenty-one 50 ml Alltech tubes is addedPolystyrene-chlorotritylchloride resin (5 g to each), dichloromethane(25 mL), and 3 equivalents of F moc-amino acids (see FIG. 3 forstructures), and 6 equivalents of diethylisopropylamine. The reactionsare shaken at room temperature for 30 minutes. The resins are drainedand washed with methanol (2×), dichloromethane (3×), andN,N-dimethylformamide (3×). The resins are then treated with 20%piperidine in N,N-dimethylformamide for 1 hour. The resins are drainedand washed with methanol (2×), dichloromethane (3×), andN,N-dimethylformamide (3×). Each resin is divided into four 25 mLAlltech tubes, and N,N-dimethylformamide (10 mL) was added. To each ofthe four different tubes was added a mixture of 5 equivalents ofBoc-Alloc amino acids (FIG. 4), 5 equivalents of HATU, and 10equivalents of diethylisopropylamine, in 10 mL of N,N-dimethylformamide.The reactions are shaken at ambient temperature for 20 hours. The resinsare drained and washed with methanol (2×), dichloromethane (3×), andN,N-dimethylformamide (3×). Each resin is treated with 0.1 equivalentsof tetrakis(triphenylphosphine)palladium(0) in N,N-dimethylformamide (10mL) for 20 hours to effect the alloc deprotection. The resins aredrained and washed with methanol (2×), dichloromethane (3×), andN,N-dimethylformamide (3×). Each resin is divided into twelve 4 mLAlltech tubes, and dichloromethane (1 mL) was added. To each of thetwelve different tubes is added a mixture of 5 equivalents of carboxylicacid (FIG. 5), 5 equivalents of HATU, and 10 equivalents ofdiethylisopropylamine, in 1 mL of N,N-dimethylformamide. The reactionsare shaken at ambient temperature for 20 hours. The resins are drainedand washed with methanol (2×), dichloromethane (3×),N,N-dimethylformamide (3×) and dichloromethane (3×). The resulting 1008tubes were treated with 90% trifluoroacetic acid in dichloromethane (0.5mL) for 3 hours, the tubes are drained into eleven 2 mL 96 deep wellplates. The solvent is removed under reduced pressure using a GeneVac.The resulting residues were dissolved in DMSO to an approximateconcentration of 100 mM and submitted for biological assay to test forcapacity to be transported via PEPT2 (e.g., using oocytes transfectedwith PEPT2 as described above).

5. Procedure for Preparing a Library to Explore for PEPT2 SpecificSubstrates

Into four 250 mL peptide vessels is addedPolystyrene-chlorotritylchloride resin (20 g to each), dichloromethane125 mL), and 3 equivalents of Fmoc-alloc-amino acids (see FIG. 6 forstructures), and 6 equivalents of diethylisopropylamine. The reactionsare shaken at room temperature for 30 minutes. The resins are drainedand washed with methanol (2×), dichloromethane (3×), andN,N-dimethylformamide (3×). The resins are then treated with 20%piperidine in N,N-dimethylformamide for 1 hour. The resins are drainedand washed with methanol (2×), dichloromethane (3×), andN,N-dimethylformamide (3×). Each resin is divided into twenty-one 25 mLAlltech tubes, and N,N-dimethylformamide (10 mL) is added. To each ofthe four different tubes is added a mixture of 5 equivalents ofBoc-amino acids (FIG. 7), 5 equivalents of HATU, and 10 equivalents ofdiethylisopropylamine, in 10 mL of N,N-dimethylformamide. The reactionsare shaken at ambient temperature for 20 hours. The resins are drainedand washed with methanol (2×), dichloromethane (3×), andN,N-dimethylformamide (3×). Each resin is treated with 0.1 equivalentsof tetrakis(triphenylphosphine)palladium(0) in N,N-dimethylformamide (10mL) for 20 hours to effect the alloc deprotection. The resins aredrained and washed with methanol (2×), dichloromethane (3×), andN,N-dimethylformamide (3×). Each resin is divided into twelve 4 mLAlltech tubes, and dichloromethane (1 mL) was added. To each of thetwelve different tubes is added a mixture of 5 equivalents of carboxylicacid (FIG. 5), 5 equivalents of HATU, and 10 equivalents ofdiethylisopropylamine, in 1 mL of N,N-dimethylformamide. The reactionsare shaken at ambient temperature for 20 hours. The resins are drainedand washed with methanol (2×), dichloromethane (3×),N,N-dimethylformamide (3×) and dichloromethane (3×). The resulting 1008tubes are treated with 90% trifluoroacetic acid in dichloromethane (0.5mL) for 3 hours, the tubes were drained into eleven 2 mL 96 deep wellplates. The solvent is removed under reduced pressure using a GeneVac.The resulting residues were dissolved in DMSO to an approximateconcentration of 100 mM and submitted for biological assay to test forcapacity to be transported via PEPT2 (e.g., using oocytes transfectedwith PEPT2 as described above).

1. A method of treatment comprising: providing a conjugate comprising anagent linked to a conjugate moiety, which conjugate has a Vmax for thePEPT2 transporter of at least 1% of the Vmax of substrate Gly-Sar forPEPT2; wherein the agent has a pharmacological activity without theconjugate moiety, and the conjugate has a greater Vmax for PEPT2 thanthe agent without the conjugate moiety, wherein the PEPT2 transporterhas the amino acid sequence of SEQ ID NO:2 or has at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:2, and the PEPT2transporter can transport Gly-Sar; orally administering the conjugate toa patient, whereby the agent exerts a pharmacological effect in thepatient.
 2. The method of claim 1, wherein the conjugate has a Vmax forthe PEPT2 transporter of at least 5% of the Vmax of substrate Gly-Sarfor PEPT2.
 3. The method of claim 1, wherein the conjugate has a Vmaxfor the PEPT2 transporter of at least 10% of the Vmax of substrateGly-Sar for PEPT2.
 4. The method of claim 1, wherein the conjugate has aVmax for the PEPT2 transporter of at least 20% of the Vmax of substrateGly-Sar for PEPT2.
 5. The method of claim 1, wherein the conjugate has aVmax for the PEPT2 transporter of at least 50% of the Vmax of substrateGly-Sar for PEPT2.
 6. The method of claim 1, wherein the conjugate has aVmax for the PEPT2 transporter of at least 100% of the Vmax of substrateGly-Sar for PEPT2.
 7. The method of claim 6, where the conjugate has aVmax for the PEPT1 transporter of less than 1% of the Vmax of substrateGly-Sar for PEPT1, wherein the PEPT1 transporter has the amino acidsequence of SEQ ID NO:1 or has at least 90% sequence identity to theamino acid sequence of SEQ ID NO:1, and the PEPT1 transporter cantransport Gly-Sar.
 8. The method of claim 1, wherein the patient is freeof a disease of the brain or kidney.
 9. The method of claim 1, whereinthe patient is free of a disease of the brain, kidney, lung or spleen.10. The method of claim 1, wherein the conjugate or the conjugate moietyhas been identified by screening a plurality of candidate substrates fortransport through the PEPT2 transporter.
 11. The method of claim 1,wherein the conjugate or the conjugate moiety has been identified byscreening a plurality of candidate substrates for transport through aPEPT1 transporter and the PEPT2 transporter, wherein the PEPT1transporter has the amino acid sequence of SEQ ID NO:1 or has at least90% sequence identity to the amino acid sequence of SEQ ID NO:1, and thePEPT1 transporter can transport Gly-Sar.
 12. The method of claim 11,wherein the plurality of candidate substrates were screened separatelyfor transport through the PEPT1 and PEPT2 transporters.
 13. The methodof claim 1, wherein the ratio of Vmax between the conjugate and Gly-Saris greater for the PEPT2 transporter than for the PEPT1 transporter,wherein the PEPT1 transporter has the amino acid sequence of SEQ ID NO:1or has at least 90% sequence identity to the amino acid sequence of SEQID NO:1, and the PEPT1 transporter can transport Gly-Sar.
 14. The methodof claim 1, wherein the ratio for the PEPT2 transporter is at leasttwice the ratio for the PEPT1 transporter, wherein the PEPT1 transporterhas the amino acid sequence of SEQ ID NO:1 or has at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:1, and the PEPT1transporter can transport Gly-Sar.
 15. The method of claim 1, whereinthe ratio for the PEPT2 transporter is at least ten times the ratio forthe PEPT1 transporter, wherein the PEPT1 transporter has the amino acidsequence of SEQ ID NO:1 or has at least 90% sequence identity to theamino acid sequence of SEQ ID NO:1, and the PEPT1 transporter cantransport Gly-Sar.
 16. The method of claim 1, wherein the ratio for thePEPT2 transporter is at least 100 times the ratio for the PEPT1transporter, wherein the PEPT1 transporter has the amino acid sequenceof SEQ ID NO:1 or has at least 90% sequence identity to the amino acidsequence of SEQ ID NO:1, and the PEPT1 transporter can transportGly-Sar.
 17. The method of claim 1, further comprising screening aplurality of conjugates or conjugate moieties to identify the conjugateor conjugate moiety. 18-62. (canceled)